In virtually all cells, changes in cytoplasmic calcium (Ca2+) concentration regulate cell functioning and signaling. Generally, a stimulus generates messenger signals that cause a release of calcium from sequestered intracellular stores (primarily found in the endoplasmic reticulum (ER)) and into the cytoplasm. The release of calcium from a sequestered intracellular store triggers calcium channels on the plasma membrane to open, which then leads to influx of calcium from the extracellular space into the cytoplasm and, in turn, into the store. The series of events between the initial release of stored calcium to the subsequent influx of calcium from the extracellular space into the cytoplasm is referred to as store-operated calcium influx (SOC influx). Sustained levels of increased cytoplasmic calcium are required for a number of physiologic responses, such as T-cell activation and differentiation.
The mechanisms that lead to calcium mobilization under physiological conditions have been studied. Stimulation of cells with a variety of physiological stimuli leads to an inositol-1,4,5-trisphosphate (InsP3)-mediated release of Ca2+ from intracellular stores which in turn triggers an influx of Ca2+ across the plasma membrane (Berridge et al., (2003) Nature Rev. Mol. Cell. Biol. 4, 517-29; Putney et al., (1986) Cell Calcium 7, 1-12; Mikoshiba et al., (2000) Sci. STKE 2000, PE1; Putney et al., (2001) J. Cell Sci. 114, 2223-29; Prakriya et al., (2003) Cell Calcium 33, 311-21; Lewis et al., (2001) Annu. Rev. Immunol. 19, 497-521; Missiaen et al., (1994) J. Biol. Chem. 269, 5817-23; Montero et al., (2001) Cell Calcium 30, 181-90).
Many of the signaling pathways leading from cell stimulation to the depletion of sequestered stores of calcium have been defined. However, the details of the subsequent pathway leading from Ca2+ store depletion to Ca2+ influx through the plasma membrane (also termed store operated Ca2+ (SOC) influx or capacitative Ca2+ entry pathway (Putney et al., (1986) Cell Calcium 7, 1-12; Mikoshiba et al., (2000) Sci. STKE 2000, PE1; Putney et al., (2001) J. Cell Sci. 114, 2223-29; Prakriya et al., (2003) Cell Calcium 33, 311-21)) have remained elusive. Recently, stromal interaction molecule (STIM)-1 (Williams et al., Biochim Biophys Acta. Apr. 1, 2002; 1596(1):131-7; Williams et al. Biochem J. Aug. 1, 2001; 357(Pt 3):673-85) was reported to play a role in the signaling processes that comprise SOC influx. (Roos et al., (2005) J Cell Biol., 169, 435-45) However, an understanding of the mechanisms through which STIM1 facilitates the calcium-based signaling processes has not been reported.
A few conventional methods are available for assessing calcium influx. These include patch clamp studies, which detect changes in calcium concentrations through detection of changes in current across a membrane. Patch clamp studies, however, are not readily adapted to high throughput assays. Other calcium influx assays involve loading cells with a dye, such as FLUO-3™, and FLUO-4™, and FURA-2™, which is sensitive to intracellular concentrations of calcium. However, these assays require the cells to be “loaded” with dye prior to, for example, evaluating the effects of an agent upon calcium signaling. Also, the dye can leak from the cells, decreasing the sensitivity of the assay as well the period of time over which an assay can be conducted. The dye can also affect calcium buffering in the cell, thus affecting the reliability and accuracy of the results. The dye can also be toxic for cells, again affecting the ability to accurately assay effects upon calcium signaling.
Accordingly, there remains a need in this art for methods to monitor changes in modulation of calcium levels in a cell and calcium signaling. The present invention addresses this need.